Subharmonic imaging (SHI) is a new nonlinear ultrasound contrast imaging mode developed by our group, which has shown promise for diagnosing breast lesions in a prospective, pilot study. Hence, the fundamental hypothesis of this project is that the angiogenesis of breast lesions can be visualized and quantified noninvasively by analyzing the vascular morphology using contrast enhanced three-dimensional (3D) grayscale SHI and that this information will improve the differentiation between benign and malignant breast lesions. A Logiq 9 ultrasound scanner (GE Healthcare, Milwaukee, WI) will be modified to perform 3D SHI and optimized for use with the ultrasound contrast microbubble agent Definity (Bristol-Myers Squibb Imaging, N Billerica, MA). Moreover, the feasibility of quantifying blood flow through SHI perfusion estimation and deriving quantitative fractal parameters describing vascular morphology via cumulative maximum intensity (CMI) SHI have been established by our group. These concepts will be expanded to 3D acquisitions and verified in vivo. The subsequent 3 years will be devoted to establish if the differentiation of benign and malignant breast masses detected by x-ray mammography can be improved upon by baseline ultrasound (grayscale and power Doppler), grayscale pulse inversion harmonic imaging or grayscale 3D SHI (as well as SHI with CMI-SHI added). This study will be conducted jointly at Thomas Jefferson University and at the University of California, San Diego. SHI data from 450 patients with breast masses, confirmed by mammography, will be acquired in this prospective, clinical trial. ROC analysis will be performed to assess whether 3D SHI can improve the sensitivity and specificity to distinguish between benign and malignant breast disease compared to mammography as well as the other 3 ultrasound modes. Furthermore, logistical regression techniques will be used to combine the 4 ultrasound imaging modes and mammography allowing all possible combinations to be compared to one another. Finally, in a subset of 85 patients (acquired at Thomas Jefferson University) the vascular morphology of the breast lesions, as established by pathology, will be compared to 3D SHI estimates of tumor neovascularity. In conclusion, this study aims to develop a novel ultrasound based method (i.e., 3D SHI) for noninvasive breast imaging and improved characterization of breast lesions in vivo. PUBLIC HEALTH RELEVANCE: Breast cancer is the most common type of cancer in women and the accurate characterization of breast masses as benign or malignant is therefore essential. However, 80 % of all breast biopsies performed today are benign and these unnecessary procedures markedly increase healthcare costs, while causing immense anxiety to the women undergoing them. This project will develop a new ultrasound imaging technique for improved diagnoses of breast masses. The long-term benefits of this study will be the development of a minimally-invasive, safe, diagnostic imaging method to accurately, re-classify breast lesions from the suspicious category to probably benign, thus avoiding unnecessary biopsies.